Biomedical subjects

M Grewe

Publications and source records attributed to M Grewe.

At least 19 recordsLinked to original sources

PubMed1997

Photocarcinogenesis and inhibition of intercellular adhesion molecule 1 expression in cells of DNA-repair-defective individuals.

Cells from patients with xeroderma pigmentosum complementation group D (XP-D) and most patients with trichothiodystrophy (TTD) are deficient in excision repair of ultraviolet (UV) radiation-induced DNA damage. Although in both syndromes this defect is based on mutations in the same gene, XPD, only XP-D, not TTD, individuals have an increased risk of skin cancer. Since the reduction in DNA repair capacity is similar in XP-D and TTD patients, it cannot account for the difference in skin cancer risk. The features of XP-D and TTD might therefore be attributable to differences in the immune response following UV-irradiation, a factor which is presumed to be important for photocarcinogenesis. We have measured the capacity of UVB radiation to inhibit expression of the immunological key molecule intercellular adhesion molecule 1 (ICAM-1) in cells from three healthy individuals in comparison to cells from three XP-D and three TTD patients. Cells from XP-D patients, but not from TTD patients, exhibited an increased susceptibility to UVB radiation-induced inhibition of ICAM-1 expression. Transfection of XP-D cells with the wild-type XPD cDNA, but not with XPC cDNA, corrected this abnormal phenotype. Thus, the skin cancer risk in DNA repair-defective individuals correlated with the susceptibility of their cells to UVB radiation-induced inhibition of ICAM-1 expression, rather than with their defect in DNA repair. The XPD protein has dual roles: in DNA repair and transcription. The transcriptional role might be important for the control of expression of immunologically relevant genes and thereby contribute to the skin cancer risk of a DNA-repair-deficient individual.

Cells, Cultured↗

PubMed1997

Induction of proinflammatory cytokines in human epidermoid carcinoma cells by in vitro ultraviolet A1 irradiation.

Ultraviolet radiation-induced expression of cytokines by keratinocytes is important for the pathogenesis of polymorphous light eruption (PLE). Because UVA1 radiation rather than UVB radiation might be a more important trigger for PLE, cells from the human epidermoid carcinoma cell line KB were exposed in vitro to UVA1 radiation (30 J/cm2) and subsequently analyzed for cytokine expression. Ultraviolet A1 irradiation induced tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 expression in KB cells at the mRNA and protein level. Upregulation of cytokine mRNA levels followed a biphasic pattern. This effect was specific for TNF alpha and IL-8 because UVA1 radiation did not induce expression of IL-1 alpha or IL-6 in these cells. Ultraviolet A1 radiation-induced expression of intercellular adhesion molecule-1 in KB cells previously was found to depend on the thiol status of these cells. Therefore, KB cells were treated with DL-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of de novo glutathione synthesis. Exposure of BSO-pretreated KB cells to UVA1 radiation significantly induced IL-1 alpha and IL-6 mRNA and protein expression. These studies demonstrate the capacity of UVA1 radiation to induce cytokine expression in human epidermoid carcinoma cells. This immunomodulatory effect may be mediated by thiol-status-dependent and -independent mechanisms.

Buthionine Sulfoximine↗

PubMed1996

Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene.

UVA radiation is the major component of the UV solar spectrum that reaches the earth, and the therapeutic application of UVA radiation is increasing in medicine. Analysis of the cellular effects of UVA radiation has revealed that exposure of human cells to UVA radiation at physiological doses leads to increased gene expression and that this UVA response is primarily mediated through the generation of singlet oxygen. In this study, the mechanisms by which UVA radiation induces transcriptional activation of the human intercellular adhesion molecule 1 (ICAM-1) were examined. UVA radiation was capable of inducing activation of the human ICAM-1 promoter and increasing ICAM-1 mRNA and protein expression. These UVA radiation effects were inhibited by singlet oxygen quenchers, augmented by enhancement of singlet oxygen life-time, and mimicked in unirradiated cells by a singlet oxygen-generating system. UVA radiation as well as singlet oxygen-induced ICAM-1 promoter activation required activation of the transcription factor AP-2. Accordingly, both stimuli activated AP-2, and deletion of the putative AP-2-binding site abrogated ICAM-1 promoter activation in this system. This study identified the AP-2 site as the UVA radiation- and singlet oxygen-responsive element of the human ICAM-1 gene. The capacity of UVA radiation and/or singlet oxygen to induce human gene expression through activation of AP-2 indicates a previously unrecognized role of this transcription factor in the mammalian stress response.

Cell Line↗

PubMed1996

Allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in chronic skin lesions of atopic dermatitis.

The majority of allergen-specific T cells derived from inhalant allergen patch test lesions in patients with atopic dermatitis were previously found to produce a restricted type-2 cytokine pattern. Recent studies, however, have revealed that in chronic eczematous skin lesions of patients with atopic dermatitis, expression of the type-1 cytokine interferon-gamma predominates. To evaluate cytokine production by allergen-specific T cells in chronic atopic dermatitis, we established house dust mite (Dermatophagoides pteronyssinus)-specific T-cell clones from the dermis of chronic skin lesions of sensitized adult patients with atopic dermatitis. Frequencies of skin-derived T cells proliferating in the presence of Dermatophagoides pteronyssinus were between one in 138 and one in 4255, indicating that only a minority of skin-infiltrating T cells are allergen specific. When these cells were analyzed for their capacity to produce interferon-gamma, the majority (71%) of these cells were found to express interferon-gamma mRNA and to secrete interferon-gamma protein, either alone or in combination with interleukin-4. Phenotypic analysis revealed that 15% of skin-infiltrating allergen-specific T cells were CD8+. No selection of Vbeta elements was detected in Dermatophagoides pteronyssinus-specific T-cell clones. These studies demonstrate that allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in lesional atopic dermatitis. The notion that the majority of allergen-specific, skin-infiltrating T cells are capable of producing interferon-gamma further supports the concept that interferon-gamma expression has major pathogenetic relevance for the chronic phase of atopic dermatitis.

Adult↗

PubMed1996

High-dose ultraviolet A1 (UVA1), but not UVA/UVB therapy, decreases IgE-binding cells in lesional skin of patients with atopic eczema.

In order to further elucidate the mechanisms by which high-dose ultraviolet A1 (UVA1) therapy leads to improvement in patients with atopic eczema, we assessed skin sections from patients before and after high-dose UVA1 therapy (n = 5) or conventional UVA/UVB therapy (n = 4) for changes in Langerhans cells and mast cells expressing the high-affinity IgE receptor Fc epsilon RI and in surface-bound IgE by histochemical and immunohistochemical techniques. The two treatment groups exhibited different patterns of changes in the number of Fc epsilon RI+, CD1a+, and mast cells within the dermis: The density of both Langerhans cells and mast cells was decreased after high-dose UVA1 therapy, but not after UVA/UVB therapy. High-dose UVA1 and UVA/UVB therapy significantly increased the number of CD1a+ cells within the epidermis, but only high-dose UVA1 reduced the relative number of IgE+ intraepidermal Langerhans cells typically found in atopic eczema. Reduction of numbers of dermal Langerhans cells and mast cells, as well as relative numbers of intraepidermal IgE+ Langerhans cells, was closely linked to significant clinical improvement by high-dose UVA1, but not UVA/UVB therapy. These studies support the notion that IgE-binding cutaneous cells are involved in the pathogenesis of atopic eczema. We propose that UVA1 radiation exerts its effects in atopic eczema, at least in part, by inhibiting Langerhans cell migration out of the epidermis and, in particular, by reducing the number of IgE-bearing Langerhans cells and mast cells in the dermis.

Cell Count↗

PubMed1996

Analysis of the production of soluble ICAM-1 molecules by human cells.

The adhesion molecule intercellular adhesion molecule-1 (ICAM-1), in addition to its membrane-associated form (mICAM-1), also exists as a soluble form (sICAM-1). sICAM-1 is capable of binding to lymphocyte function associated antigen-1 (LFA-1) molecules, and production of sICAM-1 is therefore thought to have immunomodulatory consequences. The present study, which employed normal human keratinocytes as a model for sICAM-1-producing cells, was conducted to determine the mechanism responsible for the production of sICAM-1 and to develop a strategy for specific inhibition of sICAM-1 production. Stimulation of keratinocytes with recombinant human gamma-interferon (rhIFN-gamma) induced both expression of mICAM-1 and production of sICAM-1. Western blot analysis revealed that keratinocyte-derived sICAM-1, compared to mICAM-1, had a smaller molecular size, approximately a 7-kD difference. Neither by Northern blot analysis nor by reverse-transcriptase polymerase chain reaction (RT-PCR) was any evidence for alternatively spliced ICAM-1 mRNA obtained. Addition of the protease inhibitors iodoacetamide and E-64, however, inhibited the production of sICAM-1 in a dose-dependent manner. The involvement of proteolytic cleavage in the production of sICAM-1 was corroborated in minimal peptide protection assays, in which minimal peptides covering the potential cleavage site of ICAM-1 were added to sICAM-1-producing keratinocytes. One of these peptides, ICAM cleavage inhibitory peptide (ICAM-CIP), inhibited the production of sICAM-1 without affecting mICAM-1 expression. These studies demonstrate that sICAM-1 production in human keratinocytes is due to proteolytic cleavage, and that the oligopeptide ICAM-CIP may specifically inhibit this mechanism. The capacity of ICAM-CIP to selectively prevent production of sICAM-1 may be useful for the development of novel therapeutic approaches relevant for the management of inflammation and cancer.

Amino Acid Sequence↗

PubMed1996

Interleukin-1 receptors type I and type II are differentially regulated in human keratinocytes by ultraviolet B radiation.

Since regulation of keratinocyte IL-1 receptor expression is likely to have a major impact on the biologic effects of IL-1 on epidermal cells, we examined expression, regulation, and function of IL-1R in cultured human keratinocytes. By reverse transcriptase polymerase chain reaction, human keratinocytes were shown to express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). Human keratinocyte IL-1RI mRNA expression was dependent on the differentiation state of the cell and was regulated by ultraviolet B (UVB) radiation, which initially decreased but later increased IL-1RI expression. This UVB-induced biphasic modulation of IL-1RI expression was mediated by an autocrine mechanism involving endogenously produced IL-1alpha and IL-1RI. Increased expression of IL-1RI in UVB-irradiated or IL-1alpha-stimulated keratinocytes was functionally important, because it endowed these cells with the capacity to upregulate expression of the intercellular adhesion molecule (ICAM)-1 upon IL-1alpha stimulation. Keratinocyte IL-1RII expression was regulated by UVB irradiation in an inverse manner. Significant and rapid upregulation of IL-1RII was observed within 1 h after UVB irradiation and gradually decreased to background levels within 24 h. Inverse regulation of IL-1RII versus IL-1RI was associated with opposite functions, because blocking of IL-1RII enhanced IL-1alpha effects on induction of ICAM-1 expression. These studies demonstrate that IL-1 responsiveness of UVB-irradiated keratinocytes critically depends on regulation of IL-1RI expression and that IL-1RII serves as a "decoy" receptor for IL-1, limiting rather than promoting IL-1-mediated effects.

Cells, Cultured↗

PubMed1995

Involvement of cytokines, DNA damage, and reactive oxygen intermediates in ultraviolet radiation-induced modulation of intercellular adhesion molecule-1 expression.

By virtue of its capacity to serve as a counter-receptor for lymphocyte function-associated antigen-1, intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in generation and maintenance of immunologic/inflammatory skin diseases by mediating leukocyte/keratinocyte adhesion. Ultraviolet radiation (UVR) may exert both antiinflammatory effects (e.g., UV phototherapy) and proinflammatory effects (e.g., triggering of photosensitive skin diseases) on human skin. Recent evidence indicates that UVR-induced changes of keratinocyte ICAM-1 expression constitute the molecular basis for these ambivalent properties of UVR, as UVR is able to exert two separate and even opposite effects on ICAM-1 expression. As an antiinflammatory effect, UVR may inhibit cytokine-induced up-regulation of keratinocyte ICAM-1 expression, whereas induction of ICAM-1 expression by UVR represents a proinflammatory activity. This latter effect is mediated by an autocrine mechanism involving interleukin (IL)-1 alpha. In this autocrine system, UVR exposure of human keratinocytes leads to the release of IL-1 alpha, which in turn up-regulates the expression of IL-1 receptor type 1 molecules on the keratinocyte surface, thereby increasing the sensitivity of these cells toward IL-1 alpha. As a consequence, irradiated keratinocytes are capable of responding to endogenously produced IL-1 alpha by increasing ICAM-1 expression. Modulation of keratinocyte ICAM-1 expression after UVR exposure may be observed after both short-wave UVR (UVB; 280-320 nm) and long-wave UVR (UVA1; 340-400 nm). The photobiologic mechanisms underlying UVB versus UVA1 radiation-induced ICAM-1 modulation have been found to differ. Although not completely delineated, UVB radiation-induced modulation of ICAM-1 expression appears to be mediated via the induction of DNA damage, whereas UVA1 radiation effects involve the generation of reactive oxygen intermediates.

Animals↗

PubMed1995

Analysis of the cytokine pattern expressed in situ in inhalant allergen patch test reactions of atopic dermatitis patients.

In the present study, we examined the cytokine pattern expressed in situ during the development of eczematous reactions that had been provoked in atopic dermatitis patients by patch testing with house dust mite allergen. In 24-h house dust mite allergen patch test reactions, expression of interleukin (IL)-4 mRNA and IL-2 mRNA increased, but interferon (IFN)-gamma mRNA did not, as compared with control skin. In 48-h inhalant allergen patch test reactions, however, IFN-gamma mRNA and IL-2 mRNA expression were increased above levels observed in control skin, whereas IL-4 mRNA expression was decreased below background levels. These data demonstrate that a switch from a Th2-like to a Th1-like cytokine response occurs in inhalant allergen patch tests of atopic dermatitis patients. This biphasic pattern was specific to inhalant allergen patch test reactions, as it was not observed in irritant reactions in the same patient. IFN-gamma production by T cells may be induced by the cytokine IL-12. In the present study, up-regulation of IFN-gamma mRNA expression in inhalant allergen patch test reactions was preceded by an increased expression of the p35 subunit of IL-12 mRNA. These observations suggest that increased IL-12 expression may contribute to the observed switch of the in situ cytokine secretion pattern. Further studies are necessary to determine whether a similar biphasic pattern of cytokine expression is also operative in the initiation phase of atopic eczema.

Allergens↗

PubMed1995

Interleukin-10 production by cultured human keratinocytes: regulation by ultraviolet B and ultraviolet A1 radiation.

Keratinocytes are the primary cellular target for ultraviolet radiation in human skin, and ultraviolet radiation-induced therapeutical effects may thus be mediated by keratinocyte-derived, antiinflammatory mediators. Interleukin-10 is capable of exerting antiinflammatory effects by virtue of its capacity to suppress the production of interferon-gamma. The present study therefore assessed the ability of cultured human keratinocytes to produce interleukin-10 following ultraviolet irradiation. Exposure of long-term cultured normal human keratinocytes to ultraviolet B (280-320 nm) or to ultraviolet A1 (340-400 nm) radiation caused a time- and dose-dependent induction of interleukin-10 mRNA expression and interleukin-10 protein secretion, with ultraviolet A1 radiation being the strongest stimulus. Ultraviolet radiation-induced interleukin-10 production by normal human keratinocytes was enhanced by a factor of two, when cells were cultured in high- rather than low-calcium medium. Neither addition of the ultraviolet radiation-inducible cytokines tumor necrosis factor-alpha or interleukin-1 alpha to unirradiated keratinocytes nor presence of their respective neutralizing antibodies in cultures of irradiated keratinocytes induced or inhibited interleukin-10 synthesis. Modulation of eicosanoid production by addition of prostaglandin E2 to keratinocyte cultures or disturbance of cyclooxygenase activity by indomethacin did not affect interleukin-10 production in resting or irradiated cells. These studies demonstrate that cultured human keratinocytes are capable of producing interleukin-10. Human keratinocyte interleukin-10 production is dependent on the differentiation state of the cell and induced by ultraviolet B and, in particular, ultraviolet A1 radiation exposure. This novel property of ultraviolet radiation may account at least in part for the efficacy of phototherapy in inflammatory skin diseases.

Cell Differentiation↗

PubMed1994

Lesional expression of interferon-gamma in atopic eczema.

Atopic eczema is thought to be caused by skin-infiltrating CD4 T cells of the Th1-like and/or Th2-like subtype. We assessed expression of the Th1-like cytokine, interferon-gamma, and the Th2-like cytokine, interleukin-4, in lesional atopic skin. Compared with that in normal skin, interferon-gamma and interleukin-4 mRNA expression were increased in eczematous skin lesions in 13 and 4 of 15 patients, respectively. After successful therapy of atopic dermatitis, the increased interferon-gamma mRNA expression but not the increased interleukin-4 mRNA expression was significantly down-regulated. These data indicate that in-situ expression of interferon-gamma is linked to the clinical course of atopic dermatitis.

Cell Adhesion Molecules↗

PubMed1994

Regulation of the mRNA expression for tumor necrosis factor-alpha in rat liver macrophages.

Kupffer cells are known to produce tumor necrosis factor-alpha upon stimulation with endotoxin or viruses. This tumor necrosis factor-alpha synthesis is suppressed by prostaglandin E2 or dexamethasone. Using Northern blotting and reverse transcriptase-polymerase chain reaction, it is demonstrated that endotoxin-induced tumor necrosis factor-alpha synthesis is blocked by prostaglandin E2 or dibutyryl 3':5'-cyclic adenosine monophosphate on the transcriptional level. Tumor necrosis factor-alpha itself suppressed endotoxin-evoked tumor necrosis factor-alpha mRNA expression when given in a narrow time interval with lipopolysaccharide. Interleukin-10 of human or mouse origin also inhibited the synthesis of tumor necrosis factor-alpha mRNA and protein when given more than 2 h prior to the endotoxin challenge. The suppressive effect of prostaglandin E2 lasted for more than 36 h while IL-10 blocked tumor necrosis factor-alpha production for barely 24 h. Dexamethasone reduced the endotoxin-induced tumor necrosis factor-alpha mRNA formation by approximately 50% only, although it led to nearly complete inhibition of the synthesis of the mature protein. Taken together with reverse transcriptase-polymerase chain reaction data revealing significant amounts of tumor necrosis factor-alpha mRNA in resting Kupffer cells, an additional posttranscriptional regulation of tumor necrosis factor-alpha synthesis has to be assumed. Tumor necrosis factor-alpha mRNA was not induced by interferon-gamma, interleukin-1 beta or interleukin-6 (the latter two cytokines are also synthesized by Kupffer cells), but a 24-h prestimulation of liver macrophages with interferon-gamma or phorbol ester had a modest priming effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Regulation of tumor necrosis factor-alpha-mRNA synthesis and distribution of tumor necrosis factor-alpha-mRNA synthesizing cells in rat liver during experimental endotoxemia.

Stimulated liver macrophages (Kupffer cells) are known to release a variety of inflammation-related substances, e.g. cytokines, prostanoids, and reactive oxygen intermediates. For instance, exposure of Kupffer cells in vitro to lipopolysaccharide (endotoxin) leads to a strongly enhanced synthesis of the mRNA for tumor necrosis factor-alpha, the release of the mature protein into culture media. These events are influenced by prostanoids and corticoid hormones. Kupffer cells are thought to be the only source of tumor necrosis factor-alpha within the hepatic sinusoid, but neither this cell specificity nor the regulatory influence of glucocorticoids or prostanoids has been confirmed in the intact organ. Using non-radioactive in situ hybridization, it was possible to obtain specific signals for tumor necrosis factor-alpha-mRNA in individual Kupffer cells uniformly distributed (as compared to Kupffer cells detected by immunohistochemistry) throughout the liver. Kupffer cells were the only cells in the hepatic sinusoids of lipopolysaccharide-perfused livers to express mRNA for tumor necrosis factor-alpha. Simultaneous addition of endotoxin plus dexamethasone and endotoxin and prostaglandin E2 completely suppressed the synthesis of this mRNA. Unexpectedly, the presence of mRNA for tumor necrosis factor-alpha was also detected in the intrahepatic bile duct epithelium of lipopolysaccharide-perfused livers. It is known that biologically active endotoxin is secreted via the bile ducts. These results seem to indicate that bile duct epithelium responds to inflammatory agents with synthesis of tumor necrosis factor-alpha-mRNA. One must also consider new functional aspects of bile duct epithelium in chronic inflammatory diseases, e.g. primary biliary cirrhosis, chronic sclerosing cholangitis or graft-versus-host disease.

Animals↗

PubMed1994

Post-transcriptional regulation of interleukin-6 gene expression in human keratinocytes by ultraviolet B radiation.

Exposure to increasing doses (290-315 nm) of ultraviolet (UV) B radiation is thought to profoundly affect human health. Studies on the biologic and molecular effects of UVB radiation on human skin are therefore of particular interest. There is experimental and clinical evidence to assume that UVB radiation-induced local and systemic inflammatory reactions might be mediated at least in part by UVB-induced keratinocyte-derived interleukin (IL)-6. Previously, a UVB-induced increase of steady-state levels of IL-6 mRNA was found to be a prerequisite for keratinocyte IL-6 production after UVB irradiation. The present study was aimed at addressing the question of whether in vitro UVB irradiation would increase IL-6 mRNA expression in long-term cultured, normal human keratinocytes via transcriptional or post-transcriptional mechanisms. UVB exposure (0-100 J/m2) of keratinocytes increased low baseline expression levels of IL-6 mRNA in a time- and dose-dependent manner. Using nuclear run-on assays, transcription rates of the IL-6 gene in nuclei isolated from UVB-irradiated cells were found to be essentially identical to those seen in unirradiated cells, indicating that UVB light did not lead to increased transcription of the IL-6 gene. To determine a possible post-transcriptional mechanism in UVB-induced IL-6 mRNA expression, the effects of UVB irradiation on IL-6 mRNA stability were examined. To this end irradiated and unirradiated keratinocytes were treated with actinomycin D and subjected to Northern blot analysis to calculate IL-6 mRNA half-life. As compared with unirradiated cells, IL-6 mRNA stability was increased significantly (three- to four-fold) in UVB-irradiated cells, suggesting that UVB radiation upregulates IL-6 mRNA levels in human keratinocytes by increasing the stability of IL-6 transcripts. This is the first report indicating that UVB radiation at a physiologically relevant dose may affect gene expression in human cells at a post-transcriptional level.

Blotting, Northern↗

PubMed1994

Rat liver macrophages express the 55 kDa tumor necrosis factor receptor: modulation by interferon-gamma, lipopolysaccharide and tumor necrosis factor-alpha.

Tumor necrosis factor-alpha is an important mediator of various inflammatory and immune responses. Its biological action is crucially dependent on interaction with specific cell surface receptors. Two different receptors for TNF-alpha with molecular masses of 55 and 75 kDa have been described. Here, the presence of a 55 kDa TNF receptor mRNA and the expression of its protein is demonstrated in rat liver Kupffer cells. TNF-alpha receptor was purified from detergent-solubilized rat Kupffer cells by adsorption to recombinant human TNF-alpha-Sepharose. One band of approx. 55 kDa was seen in SDS PAGE. An antibody raised against the 55 kDa TNF receptor bound specifically to the purified receptor as revealed by immunoblot analysis. Using Northern blotting, neither LPS nor TNF-alpha altered the expression of 55 kDa TNF-R mRNA, although the exposure of Kupffer cells to LPS decreased the binding of 125I-labelled TNF-alpha. Interferon-gamma clearly enhanced the level of 55 kDa TNF-R mRNA; this effect was abolished by transcriptional but not by translational inhibitors. The increase in 55 kDa TNF-R mRNA was maximal at 2-4 h of exposure of IFN-gamma. This cytokine also increased the binding of 125I-TNF-alpha to Kupffer cells. On the other hand, the amount of 55 kDa TNF-R transcripts was reduced after treatment with dexamethasone. These data suggest that in Kupffer cells the expression of the 55 kDa TNF-R is regulated at the transcriptional level.

Animals↗

PubMed1993

Analysis of the mechanism of ultraviolet (UV) B radiation-induced prostaglandin E2 synthesis by human epidermoid carcinoma cells.

Stimulation of cultured human keratinocytes with interleukin (IL)-1 alpha is known to elicit prostaglandin (PG) E2 release. Ultraviolet (UV) B radiation induces keratinocyte PGE2 and cytokine production. The present study deals with the autocrine roles of UVB-induced, keratinocyte-derived cytokines IL-1 and tumor-necrosis-factor (TNF) alpha and their corresponding receptor molecules for UVB-induced PGE2 release. In vitro exposure of transformed human keratinocytes (KB cells) induced PGE2 production five- to eightfold. This increase was inhibited by 70%, if irradiated cells were cultured in presence of monoclonal antibody (MoAb) M4, which blocks IL-1 effects by binding to the type 1 IL-1 receptor (IL-1R). In contrast, MoAb M22, which blocks the type 2 IL-1R, had no significant effects. Addition of recombinant human TNF alpha to unirradiated KB cells resulted in five- to eightfold increased PGE2 synthesis, and this increase could be mimicked by stimulation of KB cells with MoAb htr-9, which exerts TNF alpha-like bioactivity by binding to the 55-kD TNF receptor (TNFR). UVB-induced PGE2 synthesis was blocked by 50% in the presence of neutralizing anti-TNF alpha-Ab, and was completely inhibited by addition of both anti-TNF alpha-Ab and MoAb M4. To elucidate a possible regulatory intracellular step in PGE2 synthesis, specific cyclooxygenase activity in KB cells was determined. Following UVB treatment, cyclooxygenase activity increased twofold, but remained unaltered, if irradiated KB cells were cultured in the presence of anti-TNF alpha-Ab plus MoAb M4. These studies indicate that keratinocyte-derived TNF alpha and IL-1 together mediate UVB-induced PGE2 release via specific cell surface receptors, and that one intracellular mechanism is an increased prostanoid-synthesizing capacity of irradiated cells.

Antibodies, Monoclonal↗

PubMed1993

The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-alpha and by ultraviolet B radiation.

In previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR) and that its expression the important for TNF alpha-mediated upregulation of intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes. Because factors that either reduce or enhance TNFR expression are likely to have a major impact on the biological effects of TNF alpha on keratinocytes, these studies were conducted to determine the factors that regulate its expression on keratinocytes. Using reverse transcriptase polymerase chain reaction, human keratinocytes were shown to lack 75-kD TNFR expression, indicating that TNF responsiveness of human keratinocytes critically depended on regulation of 55-kD TNFR expression. Human keratinocyte 55-kD TNFR surface and mRNA expression was found to be regulated in vitro by recombinant human (rh) TNF alpha. Stimulation of keratinocytes with rhTNF alpha initially decreased, but later increased, 55-kD TNFR surface expression. This biphasic modulation of 55-kD TNFR surface expression was associated with concomitant changes in 55-kD TNFR mRNA expression. Ultraviolet B (UVB) radiation, a well-known inducer of synthesis and secretion of TNF alpha by human keratinocytes, was found to mimic TNF alpha-induced modulation of 55-kD TNFR surface and mRNA expression via a TNF alpha-mediated autocrine regulatory mechanism. Production of soluble 55-kD TNFR by human keratinocytes remained unaffected by TNF alpha stimulation or UVB irradiation. These studies provide clear evidence that membrane expression of the human 55-kD TNFR may be regulated in human keratinocytes by the ligand itself: TNF alpha. Since in previous studies UVB irradiation transiently inhibited TNF alpha-induced human keratinocyte ICAM-1 expression, it is proposed that UVB radiation-induced biphasic modulation of human keratinocyte 55-kD TNFR expression may affect the capacity of these cells to respond to TNF alpha.

Base Sequence↗

PubMed1993

Purification and quantitative analysis of nucleic acids by anion-exchange high-performance liquid chromatography.

A novel and reliable high-performance liquid chromatography (HPLC) method is described for the purification and quantification of double-stranded DNA. The nucleic acids may be obtained by polymerase chain reaction (PCR) or as restriction fragments from enzymatic cleavage; the separated products are devoid of contaminating material like agarose, ethidium bromide or non-specific DNA sequences. Because of the non-destructive nature of this HPLC procedure, the purified DNA is optimally suited for cloning experiments. The DNA separation by HPLC has major advantages when combined with reverse transcription (RT)-PCR. This is exemplified by analysis of the TNF-alpha mRNA obtained from endotoxin-elicited rat liver macrophages. If the standard procedure of Northern blotting is compared with the combination of RT-PCR and quantification of the PCR products by HPLC, it is obvious that the dynamic changes of tumor necrosis factor (TNF)-alpha mRNA synthesis are at least as precisely reflected with the RT-PCR/HPLC combination. The latter method is presented as a reliable and powerful tool for quantitative studies on gene expression.

Actins↗